Thermocellulolytic bacteria and their uses

ABSTRACT

Thermus aquaticus biovar. Nov. SK542 (FERM BP-3382) is an absolute aerobic bacteria. It grows at temperature limit of 40°-82° C. in a normal concentration medium, but its best growth is achieved at 72°-76° C. It produces protein decomposing enzymes functional at a temperature of 75°-85° C. and active in a wide pH range of 4.0-11.3, and a yellow pigment of carotenoid groups. A method for improving the quality of soil comprising applying to the soil a biologically pure culture of an absolute aerobic bacterium Thermus aquaticus biovar. nov. SK542 (FERM BP-3382) having a growth temperature limit of 40°-82° C. in a normal concentration medium, a growth optimum temperature of 72°-76° C., and producing protein decomposing enzymes functional at temperature range of 75°-85° C. and being active in a wide pH range of 4.0-11.3, and a yellow pigment of carotenoid.

This is a division, of application Ser. No. 08/256,254 filed Jun. 30,1994 which is the National Phase of PCT/JP93/00498 filed Apr. 16, 1993now U.S. Pat. No. 5,648,264.

TECHNICAL FIELD

The present invention relates to new bacteria capable of decomposingcelluloses and their usage, especially, in relation with theiragricultural usage in soil improvement. In more detail, the presentinvention relates to the bacteria useful for solubilizing naturallignin, fermenting and decomposing wood and other cellulosic materialsdifficult to be decomposed, proteinaceous substances and sludge,producing manure and compost, accelerating the maturity of humus as aproduct of decomposable biomasses, improving soil fertility and soilstructure and techniques related to those available for practical uses.

BACKGROUND TECHNIQUES

In agricultural and other industrial fields, a large amount ofhard-decomposable organic materials or hard-decomposable cellulosicsubstances, for example, sawdust, chipdust, pruner waste, bark, otherlumber industrial products such as scrap wood, chaff, rice straw, wheatstraw, hull and seed coat of beans such as soybean, red bean and peanut,coffee dregs, fallen leaves, bark, reed and mountain grass ofmiscanthus, discarded parts of shiitake (Cortinellus shiitake) and othermushrooms, and other plant materials having C/N ratio of 40-100% or moreare discarded.

Although they are composed of extremely complicated organic components,in general the essential components are about 30-75% cellulose and15-40% lignin. When both are combined, they occupy, in majority, morethan 45-90%. The nexts are hemicellulose 7-25% and those present in asmall amount such as nitrogen-containing compounds such as proteins,sugars, organic acids, alcoholic compounds. Moreover, fat, wax andessential oil are also present.

If the above-mentioned plant materials can be decomposed efficiently,the efficient uses of the discarded materials can be profitably used,and, they can be used as organic fertilizers. Moreover, as theabovementioned plant materials can be applied to soil, if they can bedecomposed efficiently, they can be a useful soil-improvement agent.

As most of plant materials contain lignin and cellulose, for efficientdecomposition of plant materials, a means for efficiently decomposingand solubilizing lignin and cellulose is essential. However, by usingexisting microorganisms and enzymes as decomposers, quick decompositionof cellulose or solubilization of lignin is extremely difficult.Furthermore, the enzymes do not act on hard proteins such as gelatin andcollagen either. As a consequence, the effective decomposition andsolubilization of natural hard-decomposable organic materials bymicroorganisms have not been successful so far.

Therefore, the present invention provides new bacteria that canexcellently ferment celluloses and solubilize lignins, a culture mixturewhose abilities are improved, a process for decomposinghard-decomposable substances, methods of soil improvement, production oforganic fertilizers, and deoderization of excreta.

DISCLOSURE OF INVENTION

Accordingly the present invention provides a bacterium Clostridiumthermocellum biovar. nov. SK522 (FERM BP-3459) capable of excellentlydecomposing cellulose, growing at temperature range of 4020 -80° C.,with best growth at 65°-72°, capable of solubilizing lignin, and anybacteria having the same properties.

The present invention further provides a bacterium Thermus aquaticusSK542 FERM BP-3382) producing yellow carotenoid pigment andprotein-decomposing enzyme which functions at pH 4.0-11.3, being activeat a temperature of 75°-85° C., growing within a temperature limit of40°-82° C. in a culture medium of regular concentration, having a growthsuitable-temperature of 72°-76° C. and having good properties, and otherbacteria possessing the same properties.

The present invention further provides a mixed culture obtained byco-culturing the above-mentioned two bacteria.

The present invention also provides a method for solubilizing lignincharacterized by acting said mixed culture on organic materialscontaining lignin.

The present invention further provides an organic material-decomposingagent comprising said mixed culture and an additive.

The present invention provides a method for improving solidcharacterized by applying said mixed culture or said organicmaterial-decomposing agent to soil.

The present invention further provides a process for production of anorganic fertilized characterized by activating said mixed culture orsaid organic material-producing organic fertilizer which is adistinctive decomposing agent to an organic material containing ligninand/or cellulose.

The present invention still more provides a method for decoratingexcreta characterized by acting said mixed culture or said organicmaterial decomposing agent to excreta.

DETAILED DESCRIPTION

First, microorganisms of the present invention are explained.

The microorganisms utilized in the present invention are new bacteria,SK522 and SK542, and strains having the same properties thereof. Thevarious properties of both bacteria are as set forth hereinafter.

Note that experiments on the following taxonomical properties andidentification are all conducted according to the Bergey's Manual ofDeterminative Bacteriology 7th edition.(1951), 8th edition (1974) andthe Bergey's Manual of Systematic Bacteriology Vol. 1 (1984) and Vol. 2(1986).

(I) Strain SK522

(Clostridium thermocellum biovar. nov. SK522, FERM BP-3459)

Taxonomical Properties

The microorganism can not grow without associating bacterium. However,since the associating bacterium can easily grow in single-separate pureculture, the microorganism and associating bacterium were cultured andseveral properties were tested in this condition.

Shape

The microorganism has a rod-like shape, but a slighly curved shape isalso present. It grows singly, but sometimes it couply grows. 0.3 to0.5×2.2 to 4.0 micron. In aged culture, they are stretched and form achain-like shape, 5.7-12.8 micron long. Peritrichous, in a dropcultureat room temperature, the movement thereof can not be seen. It formsspores having oval or circular ends, the cells are swollen and form acylindrical shape.

It is gram negative.

Cultural properties

(1) Plate Culture

The microorganism does not grow in a plate culture and otherconventional media. However, the present strain forms yellow-spottedcolonies with associating bacterium on a round-shape filter paper agarplate, as Viljoen et al. described, but the microorganism can not growalone.

(2) Slant Culture, Stab Culture

In a slant culture on agar medium of the filter paper of Viljoen et al.treated with Schweizer reagent, or in a stab culture using the samemedium, the microorganism cannot grow.

(3) Potato culture, Gelatin Culture

The microorganism can not grow by surface culture and stabbed culture.

(4) Liquid Culture

Only in Viljoen et al. medium, and media which contain cellulose, suchas aqueous cellulose peptone (less growth), cellulose bouillon, can itsgrowth be recognized. If carbon sources are substances other thancellulose, such as glucose and xylose, its ability on decomposingcellulose will be lost.

Physiological and Biochemical Properties.

1. Enzymatic Action etc.

(1) Cellulose Decomposition

The cellulose decomposing enzymes of the present bacterium areessentially a mixture of β-1, 4-glucanases. It is extracellularlysecreted, the functioning temperature being more than 80° C., heatresistant at pH more than 10.0, and some are alkali resistant. Theenzymes cut macromolecule of cellulose of micell contraction from itsend, and, glucose, cellobiose and cellooligo saccharides are formed.

The followings show a list of basic properties relating to its enzymaticsubstrates.

    ______________________________________                                            Decomposition of filter paper                                                                        FP-ase:   positive                                     Decomposition of Avicell                                                                             Avicellase:                                                                             positive                                     Decomposition of cellobiose to 2 molecules                                                           Cellobiase:                                                                             positive                                     of glucose                                                                (2) Lignin Decomposition                                                          Solubility of rice straw lignin: positive                                 (Weak, slighly weak)                                                          (3) Decomposition of protein                                                      Proteolytic enzymes:             negative                                     Peptidase:                       negative                                 (4) Hydrolysis test of starch:       slightly                                                                      positive                                 (5) Hydrolysis of hemicellulose, xylane, pectin:                                                                   negative                                 (6) Invertase, maltase:              positive                                 (7) Decomposition test of fat:       negative                                 (8) Oxidation reaction                                                            Hydroquinon reaction:            positive                                     Tyrosin reaction:                negative                                 (9) Reducing action:                 positive                                 ______________________________________                                    

2. Product Test

(1) Cellulose Fermentation

Fermentation ratio of 78-91%, cellulose is excellently fermented, andethanol, methanol, acetoaldehyde, acetate, lactate, formate, butyrate,succinate, fumarate, tartarate, gluconate, glucose, cellobiose,cellooligo saccharides, cellodextrin, large quantity of carbonate gas,hydrogen and hydrogen sulfide are produced.

(2) Products from sugars and alcohol

Acids are formed from glucose, sucrose, maltose and cellobiose.

(3) Gas generation test

From cellulose, gas is intensely produced. However, from glucose,sucrose, maltose, cellobiose, gas production is not recognized.

(4) Pepton water test

    ______________________________________                                        Ammonia:            slightly reaction                                         Indole:             positive                                                  Skatol:             positive                                                  Hydrogen sulfide:   positive                                                  ______________________________________                                    

(5) Pigment production

In general, carotenoid yellow pigment is formed.

3. Growth Condition

(1) Growth temperature

The most suitable temperature is 62°-72° C., temperature limits are40°-80° C., at lower than 40° C. it does not grow.

(2) Hydrogen ion concentration (pH)

The most suitable pH range is 6.7-8.0, pH limit is 5.6-9.6.

(3) Nitrogen source

The best source is peptone, but urea, asparagine, sodium glutamate arestill good nitrogen sources. Ammonium salt is a good inorganic nitrogensource.

(4) Carbon source

If it is continously subcultured in hydrocarbons other than cellulose,its ability of growth and fermentation will be lost.

(5) Oxygen effect

By testing at Eh=-200 to -250 mV, it is grouped into anerobic bacteria.

(6) Micronutrient requirement

Biotin, pyridoxamin, vitamin B₁₂, p-amino benzoate are required.

(7) Content of DNA's G+C

The mol % of G+C is about 38-40 (Tm).

Taxonomical Position

Having the most suitable growth temperature of 62°-72° and temperaturelimits of 40°-80° the followings are thermocellulolitic bacteria capableof excellently fermenting cellulose similar to strain SK522.

Clostridium thermocellum (Viljoen, Fred and Peterson, 1926. Jour. Agric.Sci. (London), 16, 7(1926)

Clostridium dissolvens (Bergey et al. 1925, alias Bacillus cellulosedissolvens Khouvine, 1923.: Ann. Inst. Past. 37, 711 (1923); Bergey'sManual, 2nd. Ed. (1925) p. 344.

Clostridium thermocellulaseum Enebo, 1951.: Bergey's Manual, 7th. Ed.(1957) p. 689.

Bacillus thermocellulolyticus Coolhaas. 1928.: Cent Bakt. II, 75, 101(1928), 76, 38(1929).

Bacillus thermofibrincolus Itano and Arakawa, 1929: AgriculturalChemistry 5. 816, 921 (1929); 6, 248, 257 (1930).

Agricultural Chemistry, 5. 816, 921 (1929); 6, 248, 257 (1930).

Among them, Clostridium dissoluens is most similar to the presentstrains in morphological, cultural and physio-biochemical tests exceptthe following 4 points.

(1) It is capable of solubilizing lignin weakly or very-weakly.

(2) The optimum growth temperature is 65°-72° C., growth temperaturelimit is 40°-80° C. At a temperature lower than 40° C. it does not grow

(3) It is capable of excellently fermenting cellulose, but incapable offermenting hemicellulose, xylan, pectin.

(4) The ability for solubilizing lignin is remarkable in the presence ofa microorganism belonging to the genus Thermus, and at the same time, ithas a good influence on the ability for cellulose decomposition of thepresent strain and other functions as well.

However, in the Bergey's Manual of Systematic Bacteriology Vol. 2 (1986)p. 1104 and 1141, only Clostridium thermocellum is described asthermophilic decomposing bacterium. Clostridium dissolvens and othersare considered as a subspecies or variant of C. thermocellum, orsuspected as an imperfect experimental result.

Therefore, the classification of the present strain was conducted bycomparing it with taxonomical properties of Clostridium thermocellum, atthe same time, comparing with other thermocellulolytic bacteria.

As described above, when morphological properties were compared, and theresults were summarized, the distinctively characteristical differencesof the present strain from Clostridium thermocellum and those of theabove-mentioned thermophilic cellulose-decomposing bacteria are, inaddition to the above-mentioned 4 points of differences, in thefunctions against carbohydrates, micronutrient requirement and otherproblems, and especially those related to lignin, were emphasized.

The strain has an ability to solubilize lignin. That ability can beremarkably increased if it is co-cultured with Thermus aquaticus strainSK542. Unfortunately, description relating to decomposition of lignin byClostridium thermocellum and thermocellulolytic bacteria are notcompletely available. Even if it is available, it relates to aninhibitory function concerning cellulose decomposition.

Based on such reasons, and Considering the physio-biochemical propertiesfor lignin solubilization of the present strain, the new strain wasnamed Clostridium thermocellum biovar. nov. SK522

Deposition Number

The deposition number of the present strain is FERM BP-3459. The strainis characterized as a deposited new strain based on a standard strain,belonging to Clostridium thermocellum Viljoen, Fred and Peterson (1926),having an ability for solubilizing lignin, its ability on solubilizinglignin will be remarkably increased if the strain is mix-cultured withbacterium belonging to the genus Thermus Brock and Freeze 1969. Thepresent strain is a new strain on the basis of physio-biochemicalcharacteristics covering strain SK522, and natural and artificialmutants.

Screening of Strain SK522

Using as an isolation source, soil, coastal mud, compost, human,household and animal nightsoils, enrichment culture is repeated severaltimes at 55°-65° C., and isolation is conducted with a filter paper-agarslab culture of Tetrault as suggested by Viljoen et al.

Nutrients which are contained in Vijoen et al.'s medium are enough, butit will be better if inorganic metal salts, vitamins, growth promotingfactors, for example yeast extract, are added.

    ______________________________________                                        Peptone            5.0         g                                              Calcium carbonate  saturated                                                  Sodium ammonium phosphate                                                                        2.0         g                                              Potassium phosphate                                                                              1.0         g                                              Magnesium sulphate 0.3         g                                              Calcium chloride   0.1         g                                              Iron (II) chloride Trace                                                      Cellulose (filter paper)                                                                         15.0        g                                              Well water         1000        ml                                             ______________________________________                                    

Usefulness of Strain SK522

Strain SK522 is a very excellent cellulolytic bacterium. There are twostrain types, weak and very weak ones. As they also have an ability tosolubilize lignin, when they are mix-cultured with bacterium of genusThermus, their ability especially cellulolytic ability will beremarkably increased.

Moreover, those increased ability in a mix culture may be used fordecomposing various cellulose materials. As a consequence, a method ofcompost production will be advanced, and a method of compost productionwith a high rate decomposing process can be established.

Furthermore, by using strain SK522 and SK542, soil microbial flora willbe healthily activated, the growth of useful bacteria having an abilityof buffering against environmental changes and their useful effects willbe maintained, and soil-born diseases and other problems resulted fromcontinuous cropping can be eliminated. In addition, the development oftechnology on applying them to soil and plant leaf surface,deodorization of excreta, decomposition of hard-proteinaceousesubstances, as new methods of microbial usages, can be expected.

(II) Strain SK542

(Thermus aquaticus biovar. nov. strain SK542,

Taxonomical Properties Shape

Rod-like, 0.4-0.6×3.0-5.0 micron long. It is 5 affected by mediumcondition, for example as the culture becomes aged, it is thready-like,20-130 micron long. It does not have flagellum. At room temperature, itsmovement is barely recognized. It also does not have end spores and isgram negative.

Culturing Properties

It vigorously grows with generation time of 20-50 minutes.

(1) Plate Culture

3% agar, 60° C.: yellow, comparatively minute, small circular colonies.

(2) Agar Stab Culture

Surface growth only, yellow, merely enlarged.

(3) Liquid Culture

Surface coating growth (stationary culture)

Physio-Biochemical Properties

1. Enzymatic Actions

    ______________________________________                                        (1)        Protein Decomposition                                                                           positive                                                    Proteolytic enzymes:                                                                            (strong)                                                    Gelatin hydrolysis:                                                                             positive                                                    Peptidase:        positive                                         (2)        Starch hydrolysis:                                                                              slightly                                                                      positive                                         (3)        Pectin hydrolysis:                                                                              positive                                                                      (weak)                                           (4)        Cellulose decomposition:                                                                        negative                                         (5)        Lignin decomposition:                                                                           negative                                         ______________________________________                                    

Eventhough both cellulose and lignin decompositions are negative, in amix culture with thermocellulolytic bacterium SK522, it remarkablyincreases the ability of the strain SK522 to solubilize lignin, and atthe same time, cellulose decomposition ability and other functions arepositively affected.

    ______________________________________                                         (6)      Fat decomposition:  negative                                         (7)      Invertase, maltase: positive                                         (8)      Catalase, oxidase responses:                                                                      positive                                         (9)      Hydrogen sulfide formation:                                                                       positive                                                                      (weak)                                          (10)      Sulfuric acid reduction reaction:                                                                 negative                                        ______________________________________                                    

2. Product Test

(1) Production of acid and gas from sugar and alcohol

It does not produce gas,but produces acid from glucose, galactose,maltose, lactose and glycerol.

    ______________________________________                                        (2)       Indole and skatol formation:                                                                      negative                                        (3)       Pigment production                                                  ______________________________________                                    

It produces yellow pigment-(carotinoid pigment, the muximum absorbance450 nm and small peaks at 430, 435 and 470 nm). However, in a mixculture with glucose and other sugars as carbon resources, it does notproduce pigment.

3. Growth Condition

(1) Culture concentration

Bacteria belonging to the genus Thermus are generally sensitive to theconcentration of organic substances. If the nutrient requirements arelow, low concentration should be used. However, the present strainresponds differently as even with normal concentration it can grow well.

It can also grow with more than 5% NaCl, even though the opticalconcentration is between 2.0-3.0%. In general, however, bacteriabelonging to the genus Thermus can not grow in the presence of more than2% NaCl.

It grows also with more than 3% Na-glutamate and more than 5% sucrose ormaltose. Even 2% peptone +1% yeast extract can not interfere its growth.In general, bacteria belonging to the genus Thermus grow suitably in thepresence of approximately 1% of each tryptone and yeast extract, but iftheir concentrations are increased each more than 1%, the strain can notgrow.

(2) Growth temperature

The optimum temperature is between 72°-76° C., and growth temperaturerange is 40°-82° C. It can not grow below 40° C.

(3) Hydrogen ion concentration (pH)

The most suitable pH is between 6.0-10.0. Growth pH range is 4.0.-11.0.

(4) Nitrogen source

Protein of gelatinous materials, glutamate salts, urea, and inorganicnitrogen, especially ammonium salts are frequently used.

(5) Carbon source

Glucose, sucrose, maltose, galactose, cellobiose, raffinose, stachyose,starch, glycerol, acetate, butyrate, malate, glutamate salts are used.

(6) Effects of O₂

The strain is absolutely aerobic.

(7) Micronutrient requirement

Its micronutrient requirement is high. Biotin, nicotinate amide andthiamine are important. However, for the good growth, it needs metalions such as Fe, Mn, Ca in comparatively high concentration. Inaddition, it is susceptible to the amount of those minerals.

(8) Sensitivity to antibiotics

In general, bacteria belonging to the genus Thermus and those similar toit are very sensitive to Penicillin G, Chlororamphenicol, Tetracycline,Streptomycin, Kanamycin and other antibiotics.

(9) Content of DNA's G+C

It is estimated that the mol% of G+C=68 (Tm).

Taxonomical Position

The present strain is absolute aerobic, its optimum growth temperatureis between 72°-76° C., growth temperature range is 40°-82° C., and itdoes not grow under 40° C. Because it is asporogenous, gram negativebacillus, and based on other many mycological properties, the strain isidentified to belong to the genus Thermus Brock and Freeze (1967).Moreover, if bacteria resembling to genus Thermus are considered, suchas Thermus aquaticus Brock and Freeze 1969 (Bergey's Manual, 1984, Vol.1, p. 337), Thermus thermophilous Oshima and Imahori 1974 (Int. J,Bacterial., 24, 102, 1974) and Thermus flavus Saiki, Kimura and Arima1972 (Agr. Biol. Chem., 36, 2357, 1972), the only species belonging tothe genus Thermus Brock and Freeze (1967) in the present Bergey's Manualof Systematic Bacteriology Vol. 1 (1984) p. 333 is Thermus aquaticusBrock and Freeze 1969. Moreover, as the distinctive characteristicsmentioned below are not found among the well-known species, it has beenidentified as a new strain of Thermus aquaticus, Thermus aquaticusbiovar. nov. SK542.

(1) In a mix culture with thermocellulolytic bacterium, SK522, itslignin solubilizing ability is remarkably increased. At the same time,its cellulose decomposing ability and other functions are preferablyaffected.

(2) It has a strong ability for decomposing protein in a wide pH andtemperature ranges.

(3) It is different from the species of the well-known genus Themus, asits susceptibility to the concentration of organic substances is verylow, and it grows well even in a medium with normal concentration.

(4) Its requirement on micronutrients is high, it requires vitamins andmetal ions. In addition, it is susceptible to the quantity of minerals.

(5) It produces yellow pigment (carotenoid pigment, the miximumabsorbance is 450 nm, and small peaks are observed in 430, 435 and 470nm).

Deposition Number

It was deposited as FERM BP-3382. It is identified according to astandard strain belonging to the genus Thermus aquaticus Brock andFreeze (1969). If it is mix-cultured with cellulolytic bacteria (strainSK522), the lignin solubilizing ability will be remarkably increased,and at the same time, cellulose-decomposing ability will be preferablyaffected. It is a new strain according to physiological and biochemicalproperties covering natural and artificial mutations and strain SK542.

Screening of Strain SK542

The sources for isolation of strain are humus and soil of hot-springareas of Kyushu and its neighborhood. After culturing a sample at55°-60° C., the microorganism is isolated on 3% agar plate culturemedium.

    ______________________________________                                        Isolating Medium Composition                                                  (Castenholz medium: Castenholz, R. W.; Bacterial.                             Rev., 33, 467 (1969))                                                         ______________________________________                                        Nitrilotriacetic acid   100      mg                                           CaSO.sub.4.2H.sub.2 O   60       mg                                           MgSO.sub.4.7H.sub.2 O   100      mg                                           NaCl                    8        mg                                           KNO.sub.3               103      mg                                           NaNO.sub.3              689      mg                                           Na.sub.2 HPO.sub.4      111      mg                                           FeCl.sub.3              0.28     mg                                           MnSO.sub.4.H.sub.2 O    2.2      mg                                           ZnSO.sub.4.7H.sub.2 O   0.5      mg                                           H.sub.3 BO.sub.3        0.5      mg                                           CuSO.sub.4              0.016    mg                                           Na.sub.2 MoO.sub.4.2H.sub.2 O                                                                         0.025    mg                                           (Yeast extract          5,000    mg)                                          (Triptone               5,000    mg)                                          (Sucrose                10,000   mg)                                          Total volume (with pure water, pH = about 8)                                                          1,000    ml                                           ______________________________________                                         Note: The numbers in the parentheses are modifications by the inventors. 

Usefulness of Strain SK542

The usefulness of this strain is characterized by the following threeactions and functions.

(1) In a mix culture with cellulose decomposing bacterium (strainSK522), its lignin decomposing ability is remarkably increased. Not onlyits cellulose decomposing ability is increased, but other functions arealso preferably affected.

(2) Its protein decomposing ability strongly functions in wide pH andtemperature ranges.

(3) It produces insoluble carotenoid yellow pigment. The yellow pigmentin a microbial cell becomes a water-soluble, low-molecular weightmaterial by decomposition by other microbes. It is then absorbed byplants and useful for increasing fruit qualities such as fruit taste,luster and storage life. In addition, by applying the cells of strainSK542,those basic properties are multiplied, and the growth ofheterotrophic microbes and soil-borned bacteria are stimulated.

Different from well known bacteria belonging to the genus Thermus, thepresent strain makes ecological and farming systems, varioushard-decomposable celluloic substances, scleroprotein, sewage sludge andmany organic materials useful, due to its weak susceptibility to theconcentration of organic material in addition to the above-mentionedproperties.

Moreover, this fact provides an improved productivity, overcoming,soil-borned diseases, problems related to continuous cropping,development of new techniques for deodorizing and decomposing excreta,and direct spreading to soil or leaf surface.

(III) A mix culture of Strain SK522 and SK542

1. Mass-culture of strain SK522

The strain is cultured in a previously mentioned Castenholz medium(Castenholz, 1969) under anaerobic or semi anaerobic condition, at65°-70° C. for 48-60 hours.

2. Mass-culture of strain SK542

The previously mentioned Castenholz medium (Castenholz, 1969) is used.The strain is cultured under aerobic condition by aeration or shaking,at 70°-75° C., for 24 hours.

3. Mix-culture of strains SK522 and SK542

The medium used is listed below. An appropriate amount of eachmass-culture of strain SK522 and SK542 is inoculated, and then culturedat 60°-70° C. for 24-36 hours as a mix-culture of both strains. Then,the culture is made powder using and the water content becomes to lessthan 5-7%, so that the mixture can resist the storage for more than 2years. In addition, the mass culture of both strains are taken in sameamounts, mixed well, and then used. The culture is processed into powderas the process will not change the culture properties.

The following is the medium E which is used a mix-culture medium.

    ______________________________________                                        K.sub.2 HPO.sub.4 5           kg                                              (NH.sub.4).sub.2 SO.sub.4                                                                       2           kg                                              Urea              2           kg                                              Peptone           5           kg                                              Yeast extract     5           kg                                              Filter paper (cellulose)                                                                        15          kg                                              CaCO.sub.3        saturated                                                   Water (pH about 7.0)                                                                            1000        l                                               ______________________________________                                    

4. Usefulnesses of mix-culturing of both strains

Under a condition of high temperature, mix-culturing with strain SK542of the absolute aerobic genus Themus will remarkably promote thedecomposition and fermentation of the natural hard-decomposable organicmaterials which cannot be decomposed by anaerobic strain

First, the growth of strain SK542 is supplied with activated, decomposedproteins released by strain SK522. Moreover, the decomposition andfermentation of hard-decomposable organic materials in city sewagesludge, discarded wood, compost and humus, all in solid fermentation,will certainly proceed better in aerobic condition. In addition, thegrowth of strain SK542 in the beginning stage changes condition to ahigh-temperature, anaerobic condition, so that the growth of strainSK522 becomes possible in a wild condition.

(1) Solubilization of lignin

Here, the remarkable increase of lignin solubilization by strain SK522as influenced by strain SK542 will be presented.

Hard-decomposable organic materials actually contain cellulose, ligninand other organic components which are strongly combined. Such a naturallignocellulose is actually impossible to be decomposed by strain SK522alone.

The chemical structure of lignin is not known perfectly. However,phenylpropane which has C-3 chain in a benzene ring is a basic unit.These units are converted to a three-dimensionally randomly polymerisedhigh molecular weight compound by radical reaction catalyzed byperoxidase. This structure is extremely resistant to microbialdecomposition. Most researchers and technicians know this problem verywell.

In this case, by using strain SK522, even though the process proceedsWeakly or slightly weak, lignin is expected to be solubilized. Moreover,by mix-culturing with strain SK542, for example, more than 50% of ricestraw lignin can be solubilized in approximately 10 days. This ligninsolubilization has been demonstrated for the first time.

The practical use of these important findings is that the decompositionand fermentation of humus can be accomplished quickly at hightemperatures of 50°-80° C. or more.

(2) Production of yellow pigment

Yellow pigments produced by strain SK542 and other strains belonging tothe genus Thermus are insoluble-type carotenoids. These yellow pigmentsare then decomposed by other microbes and become water-soluble, lowmolecular weight compounds. These compounds are then absorbed by plants,transported to appropriate position, and become good precursors.Moreover, along with fermented products of bacillus lysate andsecretion, not only the enlargement of root and shoot parts will bestimulated, but also the metabolism of reproductive growth of flower budformation, fruit set and fruit enlargement will be effected. These havebeen experimentally clarified in the level of molecular biology. Astheir practical usages grow, their usefulness, in fruit taste, lusterand storage properties will soon be accelerated.

(3) Complex of microbial ecological system

By applying strain SK542 having yellow pigment, the growth ofheterotrophic microbes and soil-borned microbes will be stimulated. Theimportant thing is that in the production of humus, strain SK522 as athermophilic microbe will not be directly activated. First, thenormal-temperature heterotrophic microbes are increased, and the heat offermentation is accumulated, resulting in an increase of temperature.However, individual strain of the thermocellulolytic bacteria and a fewbacteria alone do .not work in decomposition of naturallyhard-decomposable organic materials. The complex of microbial flora isnecessary. Each type of functioning bacteria is then increased.

During high temperature decomposition of hard-decomposable organicmaterials, strain SK522 is developed in the center of bacillus flora.The process of humus development does not involve in simple changes iningredient compounds. A mutual interaction among complex microbial floraand those changes is involved, and this strong relation will thencontinue efficiently.

Other efficient bacteria mixed with strains SK522 and SK542, are listedbelow.

(1) A culture of hemicellulose decomposing bacteria

Hemicellulose formes plant (cell wall) together with cellulose. They arecalled xylan, araban, dextran, mannan and galactan, dependent on sugarcomponent.

The culture of hemicellulose decomposing bacteria is prepared fromIwata's medium added with about 1% of concentrated rice straw xylan, at30°-38° C., and in a common anaerobic condition. Generally, it containsone or a mixture of more than two of Bacterium vulgatus, B. prodigiosum,B. mesentericus rubber, and Microspira agerliquefaciens.

(2) A culture of pectin decomposing bacteria

Bacteria which strongly decompose pectin are grouped into aerobic onessuch as baccilus or ethanol-acetone bacteria, and anaerobic ones such asbutylate bacteria.

According to the present invention, bacteria are prepared in Molish'smedium. The inoculation materials are soil, compost, horse manure,bagase or decayed hemp, which are incubated at 27°-35° C. for 3-5 daysas thick or thin layers, and mixed several times. The invented cultureusually contains more than one strain.

(3) A culture of soil actinomycetes

Working with soil actinomycetes (Actinomycetales Buchanan, 1917) isgenerally difficult. However, together with other microbes, theycertaintly posses a significant meaning as each organic substances,especially hard-decomposable cellulose, lignin, can be decomposed,leading to the formation of humus in soil or the production ofantibiotics for microflora control.

The culture is prepared in Waksman's medium (Waksman, 1919) byinoculating with fertile soil, compost or manure.

(4) A culture of soil fungi or yeast

The place in which soil fungi exists in a large quantity is soil whichcontains bacteria and actinomycetes. In cultivated land which containsplant root, especially in rhizosphere, they exist in a large quantityand are vigorous. They are responsible for decomposing organicsubstances of plant decays and making the soil fertile. Fungi are mainlyactive in the early stage of decomposition.

There are a lot of uncertainties in the function of soil yeast. However,there are enough numbers of yeast, and as they are able to circulate alot of vitamins and growth factors, together with other microbes, theysurely act for soil fertility.

The soil fungi and yeast culture is prepared in Csapek and Dox's medium(Csapek and Dox, 1910). They are isolated from soil, compost or manure,and then cultured.

(5) Thermophilic baccilus culture

The bacillus is thermophilic, movile and forms endogenous spores. Theyare a group of bacteria which are widely distributed naturally in hay,leaf surface and soil. Their strains are strongly heat resistant. Asthey can even grow at temperature more than 55° C., they are importantbacteria in compost production. Moreover, as they are known to secreteantibiotics, bacitracine and bacillicine, they recently have receivedattention from the field of soil-plant pathology. Trials in using thosebacteria in crop production, as a consequence, are possible, by heatinga suspension from fertile soil, compost or manure at 80° C. for 10minutes. The bacteria are then aerobically cultured in Waksman medium(Waksman, 1922) at 50°-60° C. Finally, they can be cultured as a pure ormix culture.

(6) A culture of yellow pigment producing bacteria

Yellow pigment produced by bacteria is carotenoid pigment. The bacteriaare especially found both in a large quantity and kind in maturecompost. They are also found in plant leaf surface. From those sources,they are isolated and then cultured in meat extract, peptone or yeastextract medium at 25°-35° C., under an aerobic condition. They aresimply identified from the appearing colors of yellowish green, yellow,yellowish brown or dark red. A culture of one or more than one speciesof flavobacterium, chromobacterium, pseudomonas, serratia andphototrophic bacteria is usually obtained.

The organic material decomposing process by the bacteria is enhanced bylimestone powder, dolomite powder, shell powder, powder of carapaces ofcrab and other sea shells, CaCO₃, Ca(OH)₂, perlite, permiculite,zeolite, powders of diatom earth and basic rock, pulverized peatmose andcharcoal, alone or in combination.

Micronutrients and minerals that can be added to enhance the decomposingprocess are biotin, nicotinic acid amide, thiamine, pyridoxamine,vitamin B₁₂, vitamins of paraamino benzoic acid, arginine, cystine,glutamic acid, isoleucine, leucine, lysine, methionine, phenylalanine,threonine, triphtophane, tyrosine, amino acids of barium, iron, mangan,cobalt and microminerals of calcium. As those enhancer, micronutrientsand minerals are chosen depending to existing bacteria, purposes ofusage, soil conditions, topography and climate, they have to becarefully selected.

By giving a mix culture of bacteria or adding organic materialdecomposing agents to each organic material, the decomposition can becompleted in 5-12 days at 60°-65° C. or in 7-14 days at 50°-75° C. Theprocess is best under an aerobic condition. Eventhough each organicmaterial responses differently, 2-5% of the mix culture can be added.

For soil improvement, the mix culture of bacteria or organic materialdecomposer can be applied directly to soil. Although the amount that canbe applied to the soil differs according to soil types and the amount oforganic materials, about 1,000 folds diluted substances in 200-1,000liters/ha, for example 500 liters/ha, can be used.

The present invention provides new strain SK522. In addition, a mixculture of strain SK522 and SK542 is provided and promising. Thesynergistic effects as a result of mix-culturing strains SK522 and SK542provide enhanced lignin decomposition and promote cellulosedecomposition and other processes as well.

Strain SK522 is excellent for decomposing cellulose and able to weaklyor slight-weakly solubilize lignin. Strain SK542 is absolute aerobic,thermophilic bacterium, active in a wide pH range, able to decompose andferment protein, and produces a yellow pigment of carotenoid.

The physical and chemical properties of protein decomposing enzymes ofstrain SK542 are presented in the following.

                  TABLE 1                                                         ______________________________________                                        Physical and chemical properties of                                           protein decomposing enzymes of strain                                         SK542                                                                                      Enzymes                                                          Properties     Protease A Protease B                                          ______________________________________                                        Molecular weight                                                                             39,000-32,000                                                                            28,000-23,000                                       Optimum pH      9.5-10.0  7.0-7.5                                             Usable pH range                                                                               8.0-12.0  4.0-9.5                                             Optimum temperature                                                                          75° 80°                                          Heat resistance                                                                              82° 87°                                          (50% activity after                                                           20 min.)                                                                      Substrate specificity                                                                        Haemoglobin,                                                                             In addition,                                                       insulin, Casein,                                                                         keratin,                                                           albumin    collagen,                                                                     elastin                                             Inhibitor      DFP, EDTA  DFP, EDTA                                           Activator      --         --                                                  ______________________________________                                    

Separation: Gel filtration and Sephadex G-75

Other than proteases A and B, which seem to be novel enzymes, there areseveral kinds of unstable enzymes in it.

The physical and chemical properties of pigment of strain SK542 arestated as the following.

Yellow pigment produced by the strain belongs to a carotenoid group. Itis insoluble in water but soluble in methanol, ethanol, petroleum ether,benzene, aceton, chloroform and mix solutions of aceton+methanol (7:3)and aceton+ethanol (1:1). It shows the highest peak in the infraredabsorbance at 450 nm and small peaks at 430 and 435 nm.

Moreover, by mix-culturing both new thermophilic strains, synergisticeffects can be achieved, but not additive effects, as a result of theirinteractions. Their function will be mutually increased and as a result,lignin, cellulose and protein can be decomposed rapidly.

As both strains are available for mix-culturing, fermenting anddecomposing agents for organic materials can be produced. Thatfermenting and decomposing agents, corresponding to practical usages,climate, topography and soil, contain strain SK522, SK542 and otheruseful microbes. As a consequence, their function will be increased, andnew, useful effects can be achieved.

The invented mix culture consists of the two selected bacteria,thermophilic cellulose decomposing bacteria (strain SK522) and bacillusof the genus Thermus (strain SK542), and other microbes. Correspondingto climate, topography, soil environmental conditions and cultivatedplants, some microbes can be selected and mixed.

Experiments

The followings are detail explanations on the applicable experiments ofthe invented bacteria. However, they do not mean to give limitations ontheir usages.

Example 1. Method of lignin solubilization

1. Bacteria: a large amount of mix culture of strains SK522 and SK542

a large amount of culture of strain SK522

a large amount of culture of strain SK542

Each culture is as described in the present specification.

2. Materials: the material used in this experiment was rice straw(non-glutinous type). Its analytical data is shown in Table 2.

                  TABLE 2                                                         ______________________________________                                        Analytical composition of rice straw (non-                                    glutinous)                                                                    Fat Sugars  Hemicellulose                                                                            Cellulose                                                                            Lignin Crude                                                                          protein                                                                             C/N                               ______________________________________                                        1.3 2.2     15.8       42.7   17.4    3.3   64.1                              ______________________________________                                    

Analysis was conducted according to the method of Kyusyu UniversityCrude protein=total N×6.25.

3. Experimental method

Put 10.0 g of the air-dried material into a 1000 ml Erlenmeyer flask.700 ml of medium E (non-filter paper) was carefully added, and aftersterilization, a given amount of the bacteria was inoculated, and thencultured at 65° C. for 20 days.

4. Result ##EQU1##

5. Experimental Results

                  TABLE 3                                                         ______________________________________                                        Experimental Results                                                                     Duration                                                                        2      5       10   12   15   20                                 Items        days   days    days days days days                               ______________________________________                                        Lignin solubilization                                                         (%)                                                                           Strain 522 + Strain 542                                                                    25.1   37.3    54.7 56.1 60.6 63.7                               Strain 522    3.4    6.3     7.1  7.0  8.3  8.7                               Strain 542   --     --      --   --   --   --                                 Cellulose fermentation                                                        (%)                                                                           Strain 522 + Strain 542                                                                    88.2   92.7    91.6 92.5 91.3 92.4                               Strain 522   10.6   17.6    20.7 22.3 21.8 22.0                               Strain 542   --     --      --   --   --   --                                 Protein decomposition                                                         (%)                                                                           Strain 522 + Strain 542                                                                    48.1   69.0    74.2 72.9 73.6 75.4                               Strain 522   13.2   17.4    19.5 24.7 25.4 26.3                               Strain 542   42.3   44.6    46.0 46.3 97.4 47.5                               ______________________________________                                    

As clearly shown in Table 3, in the mix culture of the thermophilicstrains SK522 and 542, each effect is not additive, but is synergistic.From the natural hard-decomposable cellulosic substances of rice straw,during 10-12 days, more than 50% lignin was solubilized, more than 90%cellulose was fermented and more than 70% protein was stronglydecomposed. Obvious differences from a single culture of strain 522 andstrain 542, were recognized.

Example 2. Effects of organic material decomposors on sawchipfermentation

1. Decomposor: 10% yeast extract (Wt/vol) and 10% peptone (wt/vol) wereadded to a mix culture of strains SK522 and SK542 to prepare adecomposor

2. Material: sawchip. A cryptomeria free, 17-20 years old, was sawn intoparticles of 3.0-1.0 mm.

                  TABLE 4                                                         ______________________________________                                        Composition of Sawchip (% dry matter)                                                      Hemicellu-               Crude                                   Fats Sugars  lose       Cellulose                                                                            Lignin protein                                                                             C/N                               ______________________________________                                        2.3  3.3     5.2        58.6   23.6   0.58 559.4                              ______________________________________                                         Analysis was conducted according to the method of Kyushu                      University. Crude protein = total N × 6.25                         

3. Experimental Method and Result Expression

Ten grams of the air-dryed material were put into a 1000 ml Erlenmeyerflask. 700 ml of medium E (no filter paper; refer to page . . . (19))were carefully added, and after pasteurizing, an appropriate amount ofthe decomposor was inoculated, and then cultured at 65° C. for 20 days.The medium was made up to compensate evaporation.

The results of lignin solubilization, cellulose fermentation and proteindecomposition were calculated as in Experiment 1.

Experimental Result

The results are shown in Table 5.

                  TABLE 5                                                         ______________________________________                                        Experimental Results                                                          Duration                                                                      Item    2 days  5 days  10 days                                                                             12 days                                                                             15 days                                                                             20 days                             ______________________________________                                        Lignin  23.5    35.1    55.6  57.3  64.0  68.7                                solubil-                                                                      ization (%)                                                                   Cellulose                                                                             84.3    90.3    92.9  91.5  92.2  91.5                                fermen-                                                                       tation (%)                                                                    Protein 43.7    54.8    60.7  64.1  68.9  71.5                                decompo-                                                                      sition (%)                                                                    ______________________________________                                         Each measurement was an average of 3-5 replications.                     

In about 10-12 days, the decomposor of organic material comprising as amain component a mix culture of strains SK522 and SK542 was able tosolubilize more than 50% lignin of the naturally hard-decomposablecellulosic substances of sawchip. As in the case of a single culture ofstrain SK522, both cellulose and protein were able to be fermented anddecomposed very steadily after those days.

Example 3. Methods of powder and granular-type decomposor and compostproduction

1. Production of decomposor

To produce powder and granular-type decomposors, a mass culture ofstrains SK522 and SK542, selected micronutrients and minerals and astabilizer of limestone powder were mixed well. The decomposor wasproduced according to standard procedure. The following example showsthe raw materials for powder-type product. If necessary, granular-typecan be produced by adding a granulator.

    ______________________________________                                        Composition of raw materials                                                  (Stabilizer: per 1000 kg of limestone powder)                                 ______________________________________                                        Mass culture of strain SK522                                                                      2          kg                                             Mass culture of strain SK542                                                                      2          kg                                             Biotin              1          g                                              Vitamin B.sub.12    1          g                                              Cystine             5          g                                              Methionine          10         g                                              Mangan              10         g                                              Cobalt              1          g                                              Yeast extract       100        g                                              Diatomaceous soil   50         kg                                             ______________________________________                                    

2. Compost production

Cryptomeria bark was used as a main raw material. The fermentor ofpowder type was added and a conventional compost production method wasused. The results are presented in the followings.

(1) Bark composition

                  TABLE 6                                                         ______________________________________                                        Bark composition (% dry weight)                                               Hemicellulose                                                                          Cellulose  Lignin  Total N Ash  C/N                                  ______________________________________                                        11.6     40.5       32.8    0.18    2.3  295                                  ______________________________________                                    

(2) Raw material Composition

    ______________________________________                                        Bark (pass through a 20 mm - sieve)                                                                 1000      kg                                            Rice bran             250       kg                                            Ammonium sulphate     2         kg                                            Lime superphosphate (added in the                                                                   20        kg                                            second turn-over)                                                             Decomposer            60        kg                                            Water                 about 60%                                               ______________________________________                                    

(3). Compost loading

While the crushed bark of 1000 kg and the decomposor were being mixedwell, water of about 60% was poured into them. The mixture was thenloaded into a 180×180 cm wood box and covered with a vinyl sheet.

(4) Manufacturing process

The compost was loaded on May 10. Being turned over 3 times, limesuperphosphate of 20 kg was added in the second turn-over, the processwas completed on July 10. As the temperature rose quickly, in the 5thday of loading the temperature reached 61° C. Thereafter, the processwas kept, running at more than 60° C. In the end of the 2nd turn-over,temperature reached the highest of 72° C. Thereafter, temperaturegradually decreased for some times, entering the end of composting. Upto July 10, the total temperature was 4,383° C. day. That means thetemperature rose 2.7°14 3.2° C. per day. Table 7 shows color changesduring turn-over periods according to Mansell's color chart.

                  TABLE 7                                                         ______________________________________                                        Color changes during the maturation of bark                                   compost                                                                       Periods            Color index                                                ______________________________________                                        First turn-over (May 20)                                                                         10 YR 6/5                                                                     (light yellowish brown)                                    Second turn-over (June 5)                                                                        10 YR 3/5 (dim brown)                                      Third turn-over (June 25)                                                                        10 YR 3/2 (dark brown)                                     After 2 months (July 10)                                                                         10 YR 2/2 (dark brown)                                     ______________________________________                                    

(5) Results

The results are shown in Table 8. By conventional methods, 3-6 months ormore are required to complete composting process, by this method,however, bark composting process was able to be completed for 60 days.For 60 days the total day-temperature reached 4,383° C day. Comparing toconventional methods, the temperature rose 2.7°-3.2° C. higher per day.Color changes during maturation indicated good results. Therefore, thedecomposor was good enough to decompose the bark. Lately, this processhas been clearly understood. All of these analytical values should meetthe standards released by Japanese Bark Compost Society or National BarkCompost Industry Union.

                  TABLE 8                                                         ______________________________________                                        Analytical values of manufactured bark compost                                Items           Analytical values                                             ______________________________________                                        Water (%)       58.6                                                          Organic matter (%)                                                                            72.4                                                          Total N (%)     1.9                                                           C/N ratio       24.4                                                          Total P.sub.2 O.sub.5 (%)                                                                     1.2                                                           Total K.sub.2 O (%)                                                                           0.4                                                           Total CaO (%)   7.3                                                           pH              6.8                                                           CEC             82 me/100 g                                                   Plant bioassay  no abnormality                                                Humusification (%)                                                                            31.2                                                          Plant bioassay: large white radish                                            Humusification: Okudashi's method was used                                    Humusification (%)                                                            ______________________________________                                    

The followings clarify the effects of mix culture or decomposers inimproving soil.

Example 4. Effects of direct application of decomposor on soilimprovements of verdue and agroforestry (1)

Objective: to study the utilities and effects of direct spreading of thedecomposor to soil for decomposing rice straw

Decomposor: the following raw materials were immediately mixed and madeinto powder or granule.

    ______________________________________                                        The proportion of raw materials                                               (stabilizer: per 1000 kg of limestone powder)                                 ______________________________________                                        Mix culture of strains SK522 and SK542                                                                4        kg                                           (as stated in III.3)                                                          Culture of soil actinomycetes                                                                         2        kg                                           (as stated in III.3.3)                                                        Culture of yellow pigment-producing                                                                   2        kg                                           microbes (as stated in III.3.6)                                               Yeast extract           200      kg                                           Micronutrients (Co, B, Cu, Fe, Zn, Mo, Mn)                                                            200      kg                                           ______________________________________                                    

1. Experimental method

(1) Place: chernozen soil of Usa-shi, Oita Prefecture

(2) Plant: tomato (strongly light responsive)

(3) Planting: January 15, seedling green house

(4) Treatments: (per 10 ares)

A: rice straw 1.5t, ammonium sulphate 7 kg, decomposor 80 kg

B: rice straw 1.5t, ammonium sulphate 7 kg

C: manure 2.0t

D: control

Note: treatments A - C were applied 30 days before transplanting

(5) Transplanting: March 20, 2500 seedlings/10 ares (2 rows of 180×50cm)

(6) Treatment arrangement: 10 seedlings per treatment, 2 replications

(7) Pruning: 5th level of topping

2. Experimental Results and Discussion

(1) Since from the appearance of first flower cluster the plantsexperienced continuously low temperature, the fruit sets of 2nd flowercluster in all treatments were inferior.

(2) Except for those of control, there were no differences in fruit setand the numbers of harvested fruits. However, fruit quality and yield oftreatment A (fermentor added) tended to be higher than those of othertreatments. Comparing to treatment C (manure treatment) the treatment Awas somewhat inferior, but total number of superior fruits of treatmentA was 97% and fruit weight was 18.6% higher than the treatment C.

(3) Changes of soil physic of treatment A provided good values in 3phase-distribution and other tests. Its humusification was much higherthan the treatment B (ammonium sulfate treatment).

In short, as the plants experienced low temperature during growthperiod, fruit sets of second flower cluster were inferior. However, theygrew well, yield, fruit quality and soil physic were good, thoseindicated good results from a direct application of the decomposer. Inother words these results demonstrated the practical uses of thedecomposer.

3. Experimental data

The data are presented in Table 9 and 10.

                                      TABLE 9                                     __________________________________________________________________________    Yield per 10 plants (May 10-July 15)                                          Superior fruits                                                                             Inferior fruits                                                                       Discarded fruits                                                                      Total                                                     weight  weight  weight  weight                                      Treatments                                                                          number                                                                            g   number                                                                            g   number                                                                            g   number                                                                            g                                           __________________________________________________________________________    A     158 42,078                                                                            57  3,084                                                                             57  3,084                                                                             287 59,837                                      (fermentor)                                                                   B     142 32,170                                                                            68  15,046                                                                            71  4,600                                                                             281 51,816                                      (ammonium                                                                     sulphate)                                                                     C     133 31,055                                                                            77  23,940                                                                            81  4,978                                                                             291 59,973                                      (manure)                                                                      D     140 32,170                                                                            60  15,582                                                                            63  3,750                                                                             263 51,502                                      (control)                                                                     Total 573 137,473                                                                           277 69,243                                                                            272 16,412                                                                            1,122                                                                             223,128                                     __________________________________________________________________________     Note:                                                                         inferior fruits were those which were cracked, malformed and more thatn       100 g.                                                                   

                  TABLE 10                                                        ______________________________________                                        Yield Percentage                                                              Superior fruits Inferior fruits                                                                           Discarded fruits                                  Treatments                                                                            number  weight  number                                                                              weight                                                                              number                                                                              weight                              ______________________________________                                        A       %       %       %     %     %     %                                   (fermentor)                                                                           55.05   70.35   25.09 24.51 19.86 5.14                                (ammonium                                                                             50.53   62.08   24.20 29.04 25.27 8.88                                sulphate)                                                                     C                                                                             (manure)                                                                              45.70   51.78   26.46 39.92 27.84 8.30                                D                                                                             (control)                                                                             53.23   62.46   22.82 30.26 23.95 7.28                                ______________________________________                                    

Example 5. Effects of direct application of decomposor on soilimprovements of verdue and agroforestry (2)

Objective: to study the utility and effects of direct spreading ofdecomposor for decomposing rice straw to soil planted with wheat.

Decomposer: the following raw materials were immediately mixed and madeinto powder or granule

    ______________________________________                                        Composition of raw materials                                                  (stabilizer: per 1000 kg of limestone powder)                                 ______________________________________                                        mix culture of strains SK522 and SK542                                                              4         kg                                            (as stated in (III).3)                                                        Culture of yellow pigment - producing                                                               2         kg                                            bacteria (as stated in (III) 3.(6))                                           Yeast extract         200       g                                             Micro mutrients (Co, B, Fe, Mn)                                                                     200       g                                             ______________________________________                                    

1. Experimental Method

(1) Place: Mitagawa machi, Kanzaki-gun, Saga Prefecture

(2) Plant: `Shirogane` wheat

(3) Treatments:

    ______________________________________                                        Treatments                                                                             Rice straw  Decomposer  N                                            ______________________________________                                        A        400 kg/10 ares                                                                            80 kg/10 ares                                                                             1.6 kg/10 ares                               B        400 kg/10 ares                                                                            80 kg/10 ares                                                                             0.8 kg/10 ares                               C        400 kg/10 ares                                                                             0 kg/10 ares                                                                             1.6 kg/10 ares                               D        0           0           0                                            ______________________________________                                         Note:                                                                         (1) The rice straw was cut into pieces of 5-6 cm long with a cutter.          (2) Before plowing, 80 kg/10 ares of cale lime were applied.             

2. Cultivation Outline

(1) Sowing period and rate: Nov. 9, 8 kg/10 acres

(2) Cultivation pattern: 4 rows, bed width 150 cm, row width 10 cm perrow

(3) Fertilizers, method (kg/10 acres)

    ______________________________________                                                            First    Mid-time                                                                             Heading-time                              PO    KO      N     fertilizer                                                                             fertilizer                                                                           fertilizer                                ______________________________________                                        10    14      14    5, 6     4.2    4.2                                       ______________________________________                                         Note:                                                                         Period of supplementary fertilizers                                           Midtime fertilizer: January 10                                                Headingtime fertilizer: March 5                                          

3. Experimental Results and Discussion

(1) Comparing to control (D), the sprouting periods of other treatmentswere 2 days late. The percent germination of control was also 7-11%higher than the other treatments.

(2) The plant heights of treatments A-C were generally low. Stalk andhead numbers of treatment A were very high. The percentages of effectivestalks of treatments A and B which were applied with the decomposer werealso high.

(3) Differences in the periods of heading and maturation amongtreatments were not recognized. The yield of treatments A and B, in termof the increase of head number, was abundant. Differences in yieldqualities among the treatments were not recognized.

As stated above, by applying raw rice straw to wheat, its germinationperiod was late, its percent germination was low and its firstelongation tended to be restrained. However, as the decomposer wasapplied and 400g of N were added per 100 kg of rice straw, N starvationwas eliminated and in the middle 10 days of February its growth wasimproved and its yield was increased.

Therefore, in applying rice straw, to avoid any restraining factors onyield increase, a direct spreading of the decomposer was proved to bebeneficial.

4. Experimental data

The data are presented in Table 11 and 12.

                                      TABLE 11                                    __________________________________________________________________________    Germination and growth                                                            Germination                                                                         Percent        No. of stalk                                                                           Effective                                   Treat-                                                                            period                                                                              Germination                                                                         Height (cm)                                                                            (per m.sup.2)                                                                          Stalk                                       ments                                                                             Month/day                                                                           %     2/10                                                                             3/1                                                                              3/24                                                                             2/10                                                                             3/1                                                                              3/24                                                                             ratio %                                     __________________________________________________________________________    A   12/5  81    10.2                                                                             13.5                                                                             31.8                                                                             232                                                                              587                                                                              844                                                                              70.6                                        B   12/5  78    10.0                                                                             13.2                                                                             31.3                                                                             195                                                                              494                                                                              822                                                                              71.4                                        C   12/5  82    10.9                                                                             12.9                                                                             33.4                                                                             214                                                                              554                                                                              816                                                                              65.9                                        D   12/3  89    11.8                                                                             13.6                                                                             35.1                                                                             200                                                                              553                                                                              791                                                                              66.2                                        __________________________________________________________________________

                                      TABLE 12                                    __________________________________________________________________________    Growth and yield                                                                             Straw                                                                             Head                                                                              No. of                                                                            Straw                                                                             Brain                                                                             Percent                                                                           1 l 100 grain                               Heading                                                                            Maturity                                                                           length                                                                            length                                                                            Head                                                                              weight                                                                            weight                                                                            yield                                                                             weight                                                                            weight                             Treatment                                                                          Month/day                                                                          Month/day                                                                          cm  cm  (per m.sup.2)                                                                     kg/a.                                                                             kg/a.                                                                             %   g   g    Grade                         __________________________________________________________________________    A    4/20 6/2  81  7.9 596 45  43.2                                                                              108 812 32.6 I                             B    4/20 6/2  82  8.3 544 45  41.2                                                                              103 809 31.6 I                             C    4/20 6/2  82  8.1 538 43  40.7                                                                              101 810 32.2 I                             D    4/20 6/2  80  7.7 524 46  40.3                                                                              100 806 31.2 I                             __________________________________________________________________________

Example 6. Effects of direct application of decomposor on soilimprovements of verdue and agroforestry (3)

Objective: prevention on the outbreak of Fusarium vine disease incucumber

Decomposer: the following raw materials were made into bowder or granule

    ______________________________________                                        Composition of raw materials                                                  (stabilizer: per 1000 kg of dolomite powder)                                  mix culture of strains SK522 and SK542                                                               4         kg                                           (as stated in (III).3)                                                        culture of soil actinomycetes                                                                        2         kg                                           (as stated in (III)3.(3))                                                     culture of yellow pigment producing bacteria                                                         2         kg                                           (as stated in (III)3.(6))                                                     yeast extract          200       g                                            Micronutrients (Co, B, Fe, Mn)                                                                       200       g                                            ______________________________________                                    

1. Experimental method

(1) Vegetable: cucumber `Asomidori`

(2) Cultivation and application: On Apr. 21 seeds were sawn in "chaffcharcoal". On Apr. 26 the seedlings were transplanted into 4.5 cmpolypots (filled with uncontaminated soil). After developing 10 leaves,on May 6 the plants were retransplanted carefully into 15 cm polypots(inoculated soil) by taking their root balls and put into the pots.

Soil used for raising the seedlings was that which has been applied withcompost of 2 ton/10 ares for several years. The soil was then sterilizedwith methyl bromide. Table 13.shows materials used. Fertilizers wereapplied on April 21. The soil was then inoculated with 5% "Fusariumcultured fung" (by volume) or April 28.

(3) Experimental arrangement: each treatment was composed of 20 plants,no repetition.

2. Experimental results and discussion

(1) Comparing to no-decomposor treatments (B - 1 to B - 4), the diseaseindex number and percent dead plants of decomposor treatments (A - 1 toA - 4) were lower and Fusarium vine disease was not developed.

                  TABLE 13                                                        ______________________________________                                        Materials and fertilizers added                                                                                             'Ni-                                             Fer-   Amm.                  gat-                                             men-   sul-  CDU-       Sul- suchi'                          Treat-           tor    phate S555  Talc phide                                                                              lime                            ments Items      (g/l)  (g/l) (g/l) (g/l)                                                                              (g/l)                                                                              (g/l)                           ______________________________________                                        A-1   Decomposor +                                                                             3.0    1.5   --    1.8  0.6  0.5                                   Ammonium                                                                      sulphate                                                                A-2   Decomposor +                                                                             3.0    --    2.0   1.8  0.6  0.5                                   CDU-S555                                                                      (trademark                                                                    name)                                                                   A-3   Decomposor +                                                                             3.0    1.5   --    1.8  0.6  3.0                                   Amm.                                                                          Sulphate +                                                                    'Nigatsuchi'                                                                  lime                                                                    A-4   Decomposor +                                                                             3.0    --    2.0   1.8  0.6  3.0                                   CDU-S555                                                                B-1   Ammonium   --     1.5   --    1.8  0.6  0.5                                   Sulphate                                                                B-2   CDU-S555   --     --    2.0   1.8  0.6  0.5                             B-3   Amm. Sul-  --     1.5   --    1.8  0.6  3.0                                   phate +                                                                       'Nigatsuchi'                                                                  lime                                                                    B-4   CDU-S555   --     --    2.0   1.8  0.6  3.0                             ______________________________________                                    

Up to the end of experiment, the percentage of dead plants in aminoniomsulphate treatment (A - 1) and CDU-S555+`Nigatsuchi` lime treatment (A -4) was 10%, their inhibition effects on the disease were high and thedevelopment of the disease was slow.

(2) Concerning plant growth and the results, decomposor-added treatments(A - 1 to A - 4) were better than any no-decomposor treatments (B - 1 toB - 4).

(3) Especially, decomposor-added treatments (A - 1 to A - 4) were veryeffective on preventing the plants from the disease. Comparing to otherdecomposor-added treatments catments (A - 2, A - 3) and non-decomposortreatments (B - 1 to B - 4), ammonium sulphate treatment (A - 1) andCDU-S555+`Nigatsuchi` lime treatment (A - 4) seemed to give betterresults.

(4) From the above results, the application of the decomposor wasclearly effective. Especially, the effects of ammonium sulphate,CDU-S555 +`Nigatsuchi` lime and their combinations on inhibiting thedisease were highly recognized.

3. Experimental data

The experimental data are presented in Table 14 and 15.

                  TABLE 14                                                        ______________________________________                                        Growth and soil chemical properties                                                                      Shoot                                                      Vine length                                                                             No. of   weight       EC                                    Treatments                                                                            (cm)      leaf     (g)    pH    (m/cm)                                ______________________________________                                        A - 1   90.3      11.4     87     6.89  1.05                                  A - 2   82.5      8.7      82     6.64  1.39                                  A - 3   85.6      10.5     84     6.96  1.21                                  A - 4   96.9      11.2     99     7.00  1.32                                  B - 1   65.2      9.9      49     6.61  1.25                                  B - 2   79.8      7.6      78     6.57  1.12                                  B - 3   77.7      7.4      70     6.83  1.16                                  B - 4   79.0      11.0     67     6.88  1.12                                  ______________________________________                                    

                  TABLE 15                                                        ______________________________________                                        Outbreak of Fusarium vine disease                                                    Disease Index Percent dead plant (%)                                   Treatments                                                                             5/14   5/29     6/10 5/14   5/29 6/10                                ______________________________________                                        A-1      0      20.5     30.0 0      10.0 10.0                                A-2      5.0    26.5     43.8 5.0    20.0 30.0                                A-3      5.0    23.0     35.0 5.0    15.0 25.0                                A-4      0      16.3     29.4 0      5.0  10.0                                B-1      15.0   25.0     80.0 15.0   15.0 50.0                                B-2      0      28.8     50.4 0      15.0 35.0                                B-3      0      28.0     48.3 0      15.0 44.0                                B-4      0      35.6     55.0 0      5.0  35.0                                ______________________________________                                         Disease index                                                                 total of individual index × 100/the highest                             index × sample number (20)                                              Index: 0 healthy                                                              1 slightly weak                                                               2 weak                                                                        3 very weak                                                                   5 dead                                                                   

Possibilities for industrial usages

As stated before, the present invention provides a useful, new strainSK522. Besides, as a mix culture of new strains SK522 and SK542 has beensuccessful, the next results are those that can be adopted to effect thesystems of microbial flora in a natural ecology, farm ecology or be madeinto production.

(1) Not only a lot of natural hard-decomposable organic materials can bedecomposed and not just simply digested, but a lot of useful materialscan be produced.

(2) As the decomposition proceeds very fast, and stable, itsproductivity can be maintained, and a method of compost production canbe developed.

(3) In addition, the complex process for decomposition ofhard-decomposable organic materials can be easily carried out in a shorttime, a formula of thermophilic fast decomposition can be established inone step.

(4) By applying both strains SK522 and SK542, the formation of soilmicrobial flora can be healthily and vigorously activated, and thegrowth and function of effective bacteria responding to changes inenvironmental factors can be maintained.

(5) At the same time, as resistances to contamination due tomiscellaneous and harmful bacteria are increased, soil-borne diseasesand problems due to continuous cropping can be overcome and congested.

(6) Referring to the preceding paragraphs, as techniques of applyinggood quality manure and compost or direct spreadings to soil and plantleaf surface are developed, characteristics of soil physic are improved,especially plant growth is activated, fertilily to support productivityis promoted.

(7) The decompositions of excreta deodorization, hard protein and sewagesludge are also effective. Those hard-decomposable celluloic substancesas N sources are effectively decomposed.

Again, as confirmed by the present invention, by mix-culturing thestrains, lignin is solubilized well, cellulose is decomposed, andprotein is strongly decomposed. Plant materials in soil or plantmaterials applied to soil and animal materials are quickly fermented anddecomposed. As consequences, soil fertility is increased or soilstructure is improved.

Reference for the 2 deposited microorganisms under Rule 13-2

Agency: Fermentation Research Institute, Agency of Industrial Scienceand Technology.

Address: Higashi 1-1-3, Tsukuba-shi, Ibaragi Prefecture, Japan.

Deposition number and date:

1. FERM BP-3382. May 1, 1991

2. FERM BP-3457. Jun. 18, 1991

I claim:
 1. A composition for use in decomposing organic materialcontaining lignin or cellulose and a culture comprising an absoluteaerobic bacterium Thermus aquaticus boivar. nov. SK542 (FERM BP-3382)having a growth temperature limit of 40°-82° C. in a normalconcentration medium, a growth optimum temperature 72°-76° C., andproducing protein decomposing enzymes functional at temperature range of75°-85° C. and being active in a wide pH range of 4.0-11.3, and a yellowpigment of carotenoid.
 2. The composition according to claim 1,comprising at least one additional component selected from the groupconsisting of vitamins, amino acids providing micronutrients andminerals in amounts effective for the growth of the bacterium.
 3. Thecomposition according to claim 1, wherein the composition is for use indecomposing cellulose and to solubilize lignin.
 4. The compositionaccording to claim 1, wherein the composition is for use in decomposingcellulose and to solubilize lignin.
 5. An absolute aerobic bacteriumThermus aquaticus boivar. nov. SK542 (FERM BP-3382) of a biologicallypure culture having a growth temperature limit of 4020 -82° C. in anormal concentration medium, a growth optimum temperature of 72°-76° C.,and producing protein decomposing enzymes functional at a temperaturerange of 75°-85° C. and being active in a wide pH range of 4.0-11.3, anda yellow pigment of carotenoid.
 6. A method for decomposing organicmaterial comprising treating at least one of a Cellulose and a lignincontaining organic material with a culture of an absolute aerobicbacterium Thermus aquaticus boivar. nov. SK542 (FERM BP-3382) capable ofassisting in the solubilizing lignin and fermenting celluloseexcellently, having a growth temperature limit of 40°-82° C. in a normalconcentration medium, a growth optimum temperature of 72°-76° C., andproducing protein decomposing enzymes functional at a temperature rangeof 75°-85° C. and being active in a wide pH range of 4.0-11.3, and ayellow pigment of carotenoid.
 7. The method according to claim 6,wherein the organic material contains chelators.
 8. The method accordingto claim 6, wherein said lignin or any bacteria having the sameproperties is solubilized.
 9. The method according to claim 6, whereinsaid organic material is excreta which is deodorized during the treatingstep.
 10. The method according to claim 6, is conducted at a temperaturehigher than 60° C.
 11. The method according to claim 6, wherein thedecomposed organic material is organic fertilizer.
 12. The method fordecomposing organic material according to claim 6, comprising treating alignin containing organic material with chelators.
 13. The methodaccording to claim 7, wherein the organic material contains eitherdesirable vitamins, micronutrients of amino acids groups or mineralsnecessary for the growth of the previously mentioned microbes, and theyellow pigment is a microbial cell which becomes a water-soluble, lowmolecular weight material by decomposition by the previously mentionedmicrobes.
 14. A method for improving the quality of soil comprisingapplying to said soil a biologically pure culture of an absolute aerobicbacterium Thermus aquaticus boivar. nov. SK542 (FERM BP-3382) having agrowth temperature limit of 40°-82° C. in a normal concentration medium,a growth optimum temperature of 72°-76° C., and producing proteindecomposing enzymes functional at temperature range of 75°-85° C. andbeing active in a wide pH range of 4.0-11.3, and a yellow pigment ofcarotenoid.
 15. The method according to claim 14, wherein said lignin issolubilized.
 16. The method according to claim 14, wherein said organicmaterial is excreta which is deodorized during the treating step. 17.The method according to claim 14, wherein the decomposed organicmaterial is organic fertilizer.
 18. The method according to claim 14,wherein the carotenoid yellow pigment produced is insolluble.
 19. Themethod according to claim 14, wherein the organic material containschelators.
 20. The method according to claim 14, wherein the organicmaterial contains either desirable vitamins, micronutrients of aminoacids groups or minerals necessary for the growth of the previouslymentioned microbes, and the yellow pigment is a microbial cell whichbecomes a water-soluble, low molecular weight material by decompositionby the previously mentioned microbes.